Proteasomes. Multicatalytic proteinase complexes.
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Animals↗
Biomedical subjects
S Wilk
Publications and source records attributed to S Wilk.
3,4-dichloroisocoumarin-induced activation of the degradation of beta-casein by the bovine pituitary multicatalytic proteinase complex.
The breakdown of beta-casein (caseinolytic activity) by the bovine pituitary multicatalytic proteinase complex (MPC) is initiated by a fourth active site different from the previously described chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide, where Cbz is benzyloxycarbonyl), trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide), and peptidylglutamyl peptide bond-hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) (Yu, B., Pereira, M. E., and Wilk, S. (1991) J. Biol. Chem. 266, 17396-17400). 3,4-Dichloroisocoumarin, a serine proteinase inhibitor, stimulated the caseinolytic activity of bovine pituitary or lens MPC, 3-18-fold under conditions under which the other three catalytic activities were inactivated. Addition of hydroxylamine to the modified enzyme did not reverse the effects of the inhibitor. A form of the proteinase exhibiting only 2-4% of control chymotrypsin-like, trypsin-like, and PGP activities degraded beta-casein with no accumulation of intermediate peptides. 3,4-Dichloroisocoumarin, by reacting with the chymotrypsin-like, trypsin-like, and/or PGP-active sites, may promote a conformational change of MPC, rendering the caseinolytic active site accessible to the substrate. Once bound to the active site, beta-casein is rapidly degraded either by the caseinolytic component itself or by a cooperative interaction with catalytic centers that are not affected by the serine proteinase inhibitor. These results imply that the caseinolytic component does not belong to the class of serine proteinases. Other proteins tested were not degraded by the 3,4-dichloroisocoumarin-treated enzyme, suggesting that the conformation of beta-casein may be more adequate for degradation by the caseinolytic component.
Amino Acid Sequence↗
Enzymatic changes of the bovine pituitary multicatalytic proteinase complex, induced by magnesium ions.
The effect of magnesium ions on the catalytic activities of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. Mg2+ markedly stimulated the breakdown of dephosphorylated beta-casein (caseinolytic activity) and the hydrolysis of Cbz-Leu-Leu-Glu-2-naphthylamide (peptidylglutamyl peptide bond hydrolyzing activity) by a 1700-fold purified preparation of MPC. Cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide (trypsin-like activity) was strongly inhibited and cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide (chymotrypsin-like activity) was weakly inhibited. Similar results were produced when enzymatic activities in the absence of Mg2+ were measured at 52 degrees C rather than at 37 degrees C. Trace protein impurities were removed by phenyl-Sepharose chromatography. This additional chromatographic step, while not changing the specific activities of hydrolysis of the three synthetic chromogenic substrates, led to a marked activation of the breakdown of dephosphorylated beta-casein. Mg2+ was not able to further stimulate the caseinolytic activities of either the phenyl-Sepharose-treated preparation or the preparation measured at 52 degrees C. Mg2+ therefore converts a "repressed" form of MPC to an "activated" form, possibly by promoting dissociation of a protein inhibitor, and may serve as a physiological regulator of this enzyme complex.
Amino Acid Sequence↗
Chemical modification of the bovine pituitary multicatalytic proteinase complex by N-acetylimidazole. Reversible activation of casein hydrolysis.
The effect of N-acetylimidazole, a mild acetylating reagent, on the catalytic activities and subunit structure of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. The trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide) and the peptidylglutamyl-peptide bond hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) of MPC were rapidly inactivated by N-acetylimidazole, whereas the chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide) was inactivated slowly. However, the hydrolysis of casein was markedly stimulated. Hydrolysis of casein by the acetylated enzyme generated a stable intermediate (21 kDa) which could be further degraded by native MPC. Treatment of acetylated MPC with hydroxylamine reversed the changes in trypsin-like and caseinolytic activities but did not restore the PGP activity. N-Acetylimidazole did not dissociate MPC but altered its migration on nondissociating gels presumably by acetylation of epsilon-amino groups of lysine residues. Hydroxylamine did not alter the gel electrophoretic appearance of the acetylated enzyme. These results indicate that acetylation of thiol or tyrosyl groups changes the trypsin-like and caseinolytic activities, and that amino group acetylation inhibits the PGP activity. Degradation of casein by MPC appears to be a sequential process with initial cleavage catalyzed by a component distinct from the chymotrypsin-like, trypsin-like, and PGP activities. The latter three components likely participate in the secondary proteolysis of the generated intermediates.
Amino Acid Sequence↗
Inhibition of pyroglutamyl peptidase II synthesis by phorbol ester in the Y-79 retinoblastoma cell.
Pyroglutamyl peptidase II (EC 3.4.19.-), a highly specific membrane-bound TRH-degrading enzyme, is inactivated in Y-79 human retinoblastoma cells by exposure to 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a biphasic manner. We have previously demonstrated a rapid decrease in pyroglutamyl peptidase II activity to 10% of the control level within 15 min, which returns to 70% of the control level by 1 h. This decrease results from enzyme phosphorylation by TPA-activated protein kinase-C. We now report a second phase of inactivation after longer exposure of cells to TPA. After 1 h, enzymatic activity slowly and progressively declined. By 7 h, only 15% of control activity remained. Cotreatment of cells with H-7, a protein kinase-C inhibitor, prevented this second phase of inactivation. Immunoblot experiments demonstrated a reduction in the amount of pyroglutamyl peptidase II in Y-79 membranes after long term exposure to TPA. Y-79 cells were labeled with [35S]methionine, and pyroglutamyl peptidase II was immunoprecipitated. A decreased incorporation of [35S]methionine paralleled the decrease in enzyme activity. These studies demonstrate that the second phase of inactivation after exposure to TPA is due to an inhibition of enzyme synthesis.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Low-dose nitrendipine in mild hypertension: a double-blind, placebo-controlled, comparative study.
The objective of this study was to determine whether 10 mg of nitrendipine once daily is adequate for treatment of mild hypertension. The study was a randomized, double-blind, multicenter, and placebo-controlled group comparison over 8 weeks (a 2-week placebo-controlled washout phase followed by a 6-week treatment phase) with measurement of blood pressure, routine laboratory tests, compliance monitoring, and recording of side effects every 2 weeks. The study subjects were 141 outpatients with mild arterial hypertension and were given one 10-mg tablet of nitrendipine or placebo once daily. The end point used was reduction in diastolic blood pressure (DBP) by at least 10 mm Hg or to less than or equal to 90 mm Hg; the responder rate at the end of the treatment phase was determined. After 6 weeks of treatment, the mean reduction in DBP was 11.8 mm Hg under nitrendipine compared with 5 mm Hg under placebo. The responder rates were 73% (53 of 73 patients) in the nitrendipine group and 26% (18 of 68 patients) in the placebo group. This difference is statistically significant. Side effects were reported by a total of 14 patients (6 in the nitrendipine group, 8 in the placebo group). There was one dropout in each group. No changes in laboratory parameters were observed. In conclusion, nitrendipine is suitable for monotherapy of mild arterial hypertension in the dosage of 10 mg once daily used in this study.
Aged↗
Probing the specificity of the bovine pituitary multicatalytic proteinase complex by inhibitors, activators, and by chemical modification.
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Amino Acid Sequence↗
Phosphorylation of the multicatalytic proteinase complex from bovine pituitaries by a copurifying cAMP-dependent protein kinase.
The multicatalytic proteinase complex (MPC) constitutes a major nonlysosomal proteolytic system that may play an important role in the processing of biologically active peptides and enzymes, as well as in intracellular metabolism. We report that at least two of its subunits of MW 28,800 (S2) and 27,000 (S3) are phosphorylated by a cAMP-dependent protein kinase (PK-A) that copurifies with the complex isolated from bovine pituitaries. The cAMP-induced phosphorylation was time dependent and inhibited by a PK-A inhibitor. Although not an integral part of the complex, PK-A activity was still present even in 1700-fold-purified and apparently homogeneous preparations by criteria of nondissociating polyacrylamide gel electrophoresis. Furthermore, we present evidence that the copurification of the two enzymes is not species or tissue specific, or dependent on a single method of purification. The copurifying kinase was stimulated 10-fold by cAMP (10 microM) and 2- to 3-fold by a peptide substrate of the MPC, but was unaffected by protein kinase C activators (calcium and a phospholipid mixture). These findings suggest that protein phosphorylation may represent a mechanism for regulating the activity of the multicatalytic proteinase complex.
Amino Acid Sequence↗
Metabolism of vasoactive peptides by plasma and purified renal aminopeptidase M.
Aminopeptidase M (AmM; EC 3.4.11.2) is a membrane-bound peptidase present on renal brush border and vascular plasma membrane. In the present study, AmM, purified from rabbit kidney cortex, produced a single immunoprecipitin line against AmM antisera, hydrolyzed alanyl-, leucyl- and arginyl-beta-naphthylamides at rates of 5.1 +/- 0.5, 3.9 +/- 0.5 and 2.6 +/- 0.3 mumol/min/mg, respectively, exhibited little or no alpha-glutamyl-, aspartyl- or glycyl-prolyl-naphthylamidase activities (less than or equal to 0.14 mumol/min/mg), and was inhibited by o-phenanthroline, amastatin (IC50 = 400 nM) and bestatin (IC50 = 6 microM). The alanyl-naphthylamidase activity of unfractionated rabbit plasma was found to be identical to purified AmM regarding relative rates of hydrolysis of alanyl-, leucyl- and arginyl-naphthylamides (100:79:42), pH optimum, and inhibition profile. In comparative studies with the purified enzyme, immunoreactive AmM accounted for essentially all of the alanyl-2-naphthylamidase activity of rabbit plasma. N-Terminal metabolism of (Met5)enkephalin by purified renal AmM was 3.92 +/- 0.69 mumol/min/mg, followed by somatostatin (1.25 mumol/min/mg), hepta(5-11)substance P (1.14 +/- 0.13 mumol/min/mg), (Asn1)angiotensin II (1.11 +/- 0.06 mumol/min/mg), angiotensin III (0.45 +/- 0.04 mumol/min/mg) and des(Asp1)-angiotensin I (0.36 +/- 0.04 mumol/min/mg). In contrast, substance P, bradykinin, (Sar1,Ala8)angiotensin II and neurokinin analogs containing modified N-termini (e.g. Ac-Arg) were resistant to hydrolysis by AmM. Peptide degradation was optimal at neutral pH and was inhibited by amastatin (IC50 = 200 nM) and bestatin (IC50 = 5 microM). Apparent Km values ranged from 15.7 +/- 0.4 microM for angiotensin III to 102 +/- 2 microM for (Met5)enkephalin. These data support a significant role for vascular and plasma AmM in the metabolism of circulating vasoactive peptides.
Amino Acid Sequence↗
Evaluation of the role of prolyl endopeptidase and pyroglutamyl peptidase I in the metabolism of LHRH and TRH in brain.
Intraneuronal peptide regulatory mechanisms are still poorly understood. The cytosolic enzymes prolyl endopeptidase (EC 3.4.21.26) and pyroglutamyl peptidase I (E.C.3.4.19.3) degrade both TRH and LHRH. Previous studies from this laboratory have not supported a role for these enzymes in the control of TRH levels. These studies have now been extended to cell and organ cultures and examine the effects of enzyme inhibition on LHRH. Exposure of dispersed hypothalamic cells or median eminences in culture to Z-Pro-Prolinal and pyroglutamyl diazomethyl ketone, specific inhibitors of prolyl endopeptidase and pyroglutamyl peptidase I respectively, did not change TRH content or recovery of released TRH. In vivo and in vitro treatment with these inhibitors did not modify the content of LHRH or recovery of this peptide upon release from several brain regions except in the olfactory bulb where an unexpected decrease in levels was observed. Olfactory bulb levels of TRH also decreased but only after prolonged in vivo inhibitor treatment. The decrease in olfactory bulb LHRH and TRH could not be accounted for by enzyme induction and is likely due to a non-specific or indirect effect of the inhibitors on the processing of these peptides. These studies demonstrate that levels of LHRH and TRH in brain are not controlled by cytosolic peptidases.
Animals↗
Inhibition of angiotensin III formation by thiol derivatives of acidic amino acids.
Angiotensin III is formed by removal of the N-terminal Asp residue of angiotensin II in a reaction catalyzed by glutamyl aminopeptidase (aminopeptidase A EC 3.4.11.7). Thiol derivatives of glutamate and aspartate in which the alpha-COOH group was replaced by -CH2SH were synthesized as inhibitors of glutamyl aminopeptidase. Glutamate thiol was a potent inhibitor of glutamyl aminopeptidase (Ki = 4 x 10(-7) M) but even more potently inhibited microsomal alanyl aminopeptidase (Ki = 2.5 x 10(-7) M). Aspartate thiol (beta-homocysteine) was a less potent but more selective inhibitor of glutamyl aminopeptidase (glutamyl aminopeptidase: Ki = 1.2 x 10(-6) M; microsomal alanyl aminopeptidase: Ki = 7.5 x 10(-6) M). Neither compound inhibited cytosolic leucyl aminopeptidase. Aspartate thiol blocked the conversion of angiotensin II to angiotensin III. These derivatives are more selective than amastatin and may be of value in studies probing the biological significance of angiotensin III.
Aminopeptidases↗
Rapid inactivation and phosphorylation of pyroglutamyl peptidase II in Y-79 human retinoblastoma cells after exposure to phorbol ester.
Pyroglutamyl peptidase II (EC 3.4.19.-), a membrane-bound metalloproteinase, is a highly specific TRH-degrading enzyme. Exposure of Y-79 human retinoblastoma cells to 12-0-tetradecanoyl phorbol 13-acetate (TPA) decreased the activity of this enzyme in a time- and concentration-dependent manner (IC50 5 x 10(-9) M). After 15 min of TPA treatment, only 10% of pyroglutamyl peptidase II activity remained. TPA treatment did not affect the activity of the cytosolic enzyme pyroglutamyl peptidase I (EC 3.4.19.3) or the membrane-bound enzyme dipeptidyl peptidase IV (EC 3.4.19.3). Pretreatment of the cells with the protein kinase C inhibitors H-7 or sphingosine prevented the inactivation of pyroglutamyl peptidase II by TPA. The time course of the TPA-mediated effect paralleled the time course of translocation and activation of protein kinase C in this cell line. Immunoblot analysis demonstrated that inactivation of pyroglutamyl peptidase II was not due to dissociation or internalization of this enzyme molecule. Incubation of TPA-activated Y-79 cell membranes with gamma-[32P]-ATP followed by immunoprecipitation revealed a time-dependent phosphorylation of a 48 kilodalton subunit of pyroglutamyl peptidase II. These studies indicate that the phorbol ester effect is mediated by protein kinase C, and reveal a mechanism of potentiation of the action of TRH at its target sites.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
[Low-temperature therapy of experimental acute pancreatitis in the rat].
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Acute Disease↗
Pyroglutamyl peptidase II inhibition specifically increases recovery of TRH released from rat brain slices.
Pyroglutamyl peptidase II (EC 3.4.19-) is a highly specific membrane-bound thyrotropin releasing hormone (TRH) degrading enzyme. To study the functional significance of pyroglutamyl peptidase II in TRH degradation, we synthesized the reversible inhibitor N-1-carboxy-2-phenylethyl (Nimbenzyl)-histidyl-beta-naphthylamide (CPHNA). CPHNA inhibited the enzyme with a Ki of 8 microM, but had no effect no TRH receptors or no prolyl endopeptidase (EC 3.4.21.26). It weakly inhibited cytosolic pyroglutamyl peptidase I (EC 3.4.19.3). CPHNA at a concentration of 10(-4) M increased both the basal and potassium stimulated recovery of TRH released from hypothalamic slices by approximately two-fold. An even higher recovery was observed in slices from brain regions with relatively high levels of pyroglutamyl peptidase II. CPHNA had no effect on the basal recovery of gamma-aminobutyric acid or Met-enkephalin released from brain slices but decreased the potassium stimulated recovery of both Metenkephalin and gamma-aminobutyric acid. These data further support the involvement of pyroglutamyl peptidase II in the extracellular inactivation of brain TRH.
Aminopeptidases↗
Regulation of thyrotropin releasing hormone degrading enzymes in rat brain and pituitary by L-3,5,3'-triiodothyronine.
The effect of treatment with L-3,5,3'-triiodothyronine (T3) on the levels of pyroglutamyl peptidase I and pyroglutamyl peptidase II in rat brain regions, pituitary, and serum was studied. Pyroglutamyl peptidase I cleaves pyroglutamyl peptides such as thyrotropin releasing hormone (TRH), luteinizing hormone releasing hormone, neurotensin, and bombesin, whereas pyroglutamyl peptidase II appears to be specific for TRH. Acute administration of T3 did not affect pyroglutamyl peptidase I in any of the regions studied, whereas pyroglutamyl peptidase II was significantly elevated in frontal cortex and pituitary. Treatment with T3 for 10 or 14 days significantly elevated pyroglutamyl peptidase I in pituitary, hypothalamus, olfactory bulb, hippocampus, and thalamus. Chronic T3 treatment elevated pyroglutamyl peptidase II in frontal cortex and in serum. These studies demonstrate regulation of neuropeptide degrading enzymes by thyroid hormones in vivo. This regulation may play a role in the negative feedback control of thyroid status by T3.
Aminopeptidases↗
Inhibitors of TRH-degrading enzymes.
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Aminopeptidases↗
Sodium butyrate induces pyroglutamyl peptidase I and decreases thyrotropin-releasing hormone receptors in GH3 cells.
The effect of sodium butyrate treatment on TRH-degrading enzymes and TRH receptors in GH3 cells was investigated. The specific activity of pyroglutamyl peptidase I (EC 3.4.19.3) was increased by exposure to sodium butyrate in a time- and concentration-dependent manner, whereas the specific activity of prolyl endopeptidase (EC 3.4.21.26) was unchanged. The maximal effect occurred at a concentration of 1 mM sodium butyrate and 16 h after exposure. The increase was reversible upon removal of sodium butyrate from the cell culture. Cycloheximide totally blocked the stimulation, indicating that the increase was due to new protein synthesis. Sodium butyrate had no effect on pyroglutamyl peptidase I activity in the AtT-20 cell line. [methyl-3H]TRH binding to intact GH3 cells was reduced to 70% of the control value when cells were exposed to 1 mM sodium butyrate for 8 h. A maximal decrease in binding to 40% of the control value occurred after 16 h of exposure. The Kd of [methyl-3H]TRH binding was not changed. Sodium butyrate altered GH3 cell morphology, but the morphological changes occurred after alterations of pyroglutamyl peptidase I activity and [methyl-3H]TRH-binding sites. Other agents known to alter GH3 cell morphology had no effect on pyroglutamyl peptidase I activity. These results indicate that sodium butyrate can in some respects mimic the action of T3 on GH3 cells. Moreover, they provide further evidence that the activity of pyroglutamyl peptidase I, but not prolyl endopeptidase, is subject to regulation in the GH3 cell.
Aminopeptidases↗
Occurrence of pyroglutamyl peptidase II, a specific TRH degrading enzyme in rabbit retinal membranes and in human retinoblastoma cells.
Pyroglutamyl peptidase II, a highly specific thyrotropin releasing hormone (TRH)-degrading enzyme is found in highest concentration in brain where it is localized to synaptic membranes. Retina contains relatively high concentrations of both immunoreactive TRH and TRH receptors. We report that the specific activity of pyroglutamyl peptidase II in rabbit retinal membranes exceeds that of all non-CNS tissues thus far studied. Nine clonal cell lines were screened for this enzymatic activity. The specific activity of pyroglutamyl peptidase II in Y79 retinoblastoma cells was greater than the highest activity found in other cell lines by approximately one order of magnitude. These studies further support a functional relationship between pyroglutamyl peptidase II and TRH and identify a cell line suitable for studies on the regulation of this enzyme.
Aminopeptidases↗